Summary: Aim of this project is to genetically engineer T lymphocytes to be directed at and exert effector function against tumors expressing heterodimers of the epidermal growth factor receptor (EGFR)/ErbB family of surface molecules. To achieve this goal, we will engineer transmembrane chimeric molecules with the extracellular region expressing the receptor-binding domains of the EGFR/ErbB ligands, heregulins (HRG), and the cytoplasmic domains of the zeta chain of the T cell receptor (TCR). The working hypothesis under consideration is that the EGF-like binding domain of these HRG/zeta chain chimeras will mediate the recognition of the ErbB receptors expressed on the surface of tumor cells, thus promoting recruitment of the transduced T lymphocytes at the tumor site. HRG is a convenient molecule of simple engineering with potential application for use in animals and humans. Other binding molecules, however, could be used for the extracellular component of the chimera. The cytoplasmic domain of the chimeric protein, derived from the TCR zeta chain, encompasses the primary signal transducer of the TCR. Throughout its immuno-specific tyrosine activation motifs (ITAM's), this chain is expected to promote the activation of downstream signaling and to induce a specific cytotoxic immune response in situ. A requirement for co-stimulatory molecules is anticipated: To this end, the engineering of chimeric constructs expressing the HRG binding domains and cytoplasmic domains of the T cell co-stimulatory molecule, CD28, is also being undertaken. HRG/zeta and HRG/CD28 chimeric molecules are currently being engineered and will be ectopically expressed by in vitro (ex vivo) transduction on the surface of T cell lines and primary isolated T lymphocytes. Transfected T cells will be characterized biochemically and functionally. Consideration has been given to optimizing expression and functionality of the chimeric constructs by using different HRG isoforms, extracellular hinges (spacers), and transmembrane domains. An in vitro model has being established to measure the ability of the chimeric molecules to activate the cascade of events that normally follows the interaction of the TCR with the antigen. Tumors expressing ErbB molecules will be used as targets. Biochemical parameters of signal transduction, activation markers, lymphokine secretion, proliferation, cytotoxic function in response to ErbB-positive tumor challenges have been examined in vitro. Retroviral vectors have been engineered for in vivo applications. A animal model for pre-clinical testing is being developed for fine-tuning of response characteristics, such as the engineering intracellular zeta chains expressing different ITAM number and/or types.